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Calcium association with anionic lipids: Modification by uncharged co-constituents
Colloids and Surfaces, 14 (1985) Elsevier Science Publishers B.V.,
303-307 Amsterdam
303 -
Printed
in The Netherlands
CALCIUM ASSOCIATION WITH ANIONIC LIPIDS: MODIFICATION BY UNCHARGED CO-CONSTITUENTS
D.P. GREGORY*,
J. MINGINS
and A.L.
SMITH
Unilever Research Port Sunlight Laboratory, Merseyside L63 3JW (United Kingdom) (Received
6 October
1983;
accepted
Quarry
in final form
Road
25 July
East, Bebington,
Wirral,
1984)
ABSTRACT Calcium ion association to surfactant and phospholipid species has been examined using radio-counting, electrokinetic and ion-displacement experiments. Pure and mixed insoluble, charged monolayers spread at an air/water interface were used as a model membrane system. Over the range of primary charge densities examined (0.5-2.5 nm* per charge) for long-chain sulphonate monolayers, the net wall-charge density in the presence of calcium chloride is compounded of the primary head-group charge and associated Ca*+counterions with co-ions playing an insignificant part. The strongly associated Ca2+ is displaced with difficulty by Na’, is isomorphously displaced by Mg*+ and readily displaced by Fe3+. No calcium association to pure monolayers of the uncharged molecules was observed. By spreading mixed monolayers of varying mole fraction and choosing areas of compression, the primary charge density can be held constant whilst at the same time the local monolayer environment is altered. By such means the binding of Cal+ to long-chain sulphonate is studied in the presence of various surface densities of nonionic, zwitterionic or cationic monolayer molecules. Distinct differences in the form of the binding in the various binary mixtures are observed. Similarly, differences had been seen in our earlier work where the anionic species was phosphatidylserine and was mixed with either cholesterol or phosphatidylcholine. Simple models to interpret these findings will be proposed and the possible implications discussed.
INTRODUCTION
The association of calcium ions with anionic lipids is of importance in both surfactant and biological systems. Divalent counterions adsorb strongly at ionic species though there are uncertainties both in the statistics for site binding and the distribution of sites. Examination of the literature reveals no detailed investigation of the number of sites needed nor the influence of the distribution of the sites on the binding. In biological systems, the role of calcium-phosphatidylserine (PS) interaction has prompted studies in model membrane systems. Whilst there have been many studies of the binding of *To
whom
correspondence
0166-6622/85/$03,30
should
0 1985
be addressed.
Elsevier
Science
Publishers
B.V.
304
Ca” to charged tems by charged niques.
PS ]I, 21, the possible modification of the Ca2’ binding by unspecies has received less attention. This paper deals with such sysexamining mixed insoluble monolayers, comprising anionic and unspecies, by radiotracer, surface-pressure and electrokinetic tech-
EXPERIMENTAL
The lipids used were the same samples as described elsewhere [3--51 but were of the highest purity available and in general further purified by HPLC. Inorganic chemicals and water were also as described elsewhere [6]. The lipids were spread to form insoluble monolayers at the air/water interface in a 25 X 6 X 1 cm PTFE trough. Where mixtures were used they were premixed before spreading. The subphase contained 45Ca2’ (from Radiochemical Centre, Amersham, U.K.) diluted with non-radioactive CaCl, to give a final calcium concentration of 1.0 X 10e4 M. The temperature was 20 + 1” C and the pH was approximately 5.5. Surface-associated radioactivity was measured by a modification of the method of Aniansson [7] using an Actigraph-3 gas-flow counting head linked to an ES1 Nuclear ratemeter (model 5350) whose output drove a pen recorder. The increase in radiocount rate relative to background following spreading of a monolayer could be converted to surface-associated Ca’+ by applying a factor obtained by added small known volumes of the subphase CaCl, solution to a glass plate of known area, followed by drying and counting. During radiocounting, monolayer surface pressures were monitored by the Wilhelmy plate method with zero contact angle, using a Beckman LM 600 microbalance, and the area was varied by compression. Electrokinetic measurements were made either underneath a spread lipid monolayer [8] or by preparing mixed particles of PS with cholesterol [3] and measuring their electrophoretic mobilities using a Rank Mk II apparatus with a four-electrode cylindrical cell (Rank Bras, Bottisham, Cambridge, U.K.). The temperature and pH were the same as in the monolayer experiments. RESULTS
AND DISCUSSION
Figure 1 shows surface-pressure/area (n/A) isotherms for monolayers of pure octadecylsulphonate (C,,SO;) and mixtures of this with octadecanol. It can be seen that there is a condensing effect of the octadecanol on the isotherm as previously reported for similar mixtures [9]. The isotherms for each mole ratio from 100/O down to 34/66 C,, SO;/octadecanol are in all cases more condensed than if the substrate were 10-l M NaCl indicating a greater binding of Ca” over Na”. To quantify this Ca” contribution, radiolabelling of the Ca2’ was used. However, because of the long range of the isotope used (45Ca) the mea-
305
sured calcium uptake arises from both bound and diffuse counterions and will be referred to here as associated calcium. For pure sulphonate monolayers, the associated calcium is linearly dependent on the surface concentration of the lipid over the range examined, namely 0.5-2.5 nm* per charge. Denoting the degree of calcium association as Pcaz + and defining it as the surface number density of Ca*’ divided by the surface density of sulphonate, then the data yield a figure of 0.48 for pca2+ over the range examined. This is close to the expected value of 0.5 for a fully bound calcium with a negligible co-ion contribution at such high wall charges. The effect of various added electrolytes on calcium displacement was examined by adding appropriate amounts of stock electrolyte solution to the substrate. Sodium is effective in reducing calcium association only when present at very high levels (> 10“ M), a result again indicative of high specificity of calcium against sodium in agreement with previous findings [lo, 111. In these experiments, magnesium was isomorphous with calcium but ferric ions at the 10e4 M level reduced the calcium association to almost zero. The effect of a second component on the calcium association was examined by incorporating other insoluble materials into the anionic mono-
02
04
03 A /nd
05
06
molecules’
Fig. 1. n/A isotherms for mixtures of C,,SO;/C,,OH at the air-10-4 M CaCl, interface. pH = 5.5, 2’ = 20°C. The mole fraction of C,,SO; is displayed on all the curves (X 100) together with that of the C,,OH on the more expanded curves. The C,,OH label is missing from the more crowded condensed curves but can be readily calculated by difference.
306
layer and comparing the counts at the same surface number density of C,,SO; for various surface mole ratios achieved by means of different degrees of compression. The monolayers were pre-mixed in a common solvent before spreading. Also all calcium measurements were done below the equilibrium spreading pressure of the mixture under investigation. If the second component is an equimolar amount of a cationic species, then the calcium association is reduced to zero. The radiocounting results for sulphonate mixed with octadecanol are shown in Fig. 2. pcaz+ is again as defined for pure sulphonate monolayer. No calcium association was seen for pure octadecanol monolayers. As the amount of spacer material (alcohol) is increased pcaz+ remains substantially constant (there is evidence for a slight decrease of unknown origin as in some previous work [12, 131) up to about 60% “spacer” content, after which there is an increase consistent with a charge reversal occurring in the surface region. Similar behaviour is found for other spacer materials, e.g., methylstearate and in the related phosphatidylserine/ cholesterol system [3] . If the spacer material is zwitterionic, i.e. uncharged but possessing separated positive and negative charges, then it is found that there is no detectable effect on the calcium association either to sulphonate or PS monolayers. These results suggest that calcium binding to the anionic species in the presence of zwitterion permits participation of the anionic moiety of the zwitterion in balancing the double cationic charge of the calcium whereas,
---t
I
I 025 Mole
050 fraction
I 075 %PH
Fig. 2. Calcium binding ratio @Ca z+ as a function of mole fraction monolayers at the air-lO_“ M CaCl, interface. pH = 5.5, T = 20°C.
of C,,OH
in C,,SO;
307
with alcohol as the spacer molecule, unpaired anionic sites might function separately as calcium binding sites leading to a net positive charge of the monolayer. As more spacer is added to the mixture, the probability of anionic molecules being isolated will increase. A simple statistical model can be used to predict the calcium-associated behaviour with varying spacer content and this shows a general agreement between theory and experiment [4, 51. Some implications of a net positive charge in mixed films containing high levels of nonionic monolayer molecules are: (i) There will be an increased electrical contribution to the surface pressure which should expand the n/A isotherm. Close examination of Fig. 1 shows such an expansion with increasing octadecanol content which is not seen on 10-l M NaCl. (ii) In the absence of spacer, calcium is associated very strongly with the film even when Na’ is present at 10e2M. A film which is net positive would be expected to reduce its free energy by exchange with Na’ to reduce the net wall charge. Radiocounting on such films shows Na’ to displace Ca2+ more readily from these high spacer content systems. (iii) If there is a net positive charge at the surface then this should be reflected in the electrokinetic properties of the surface. In the system examined here, the spacer itself has (for unknown reasons) a substantial negative zeta-potential and this contribution needs to be apportioned to the mixture to extract the anionic/Ca2’ potential. Two systems were examined, sulphonate/octadecanol and PS/cholesterol and in neither case was a net positive potential observed although the zeta-potential is numerically lower in the mixture than in the pure state. Surface-potential measurements may aid in the understanding of the electrical properties of these systems. The model developed (and reported in detail elsewhere [4, 51) to accommodate the binding behaviour requires the gradual isolation of the C,,SO; anions from each other with increasing mole fraction of octadecanol so that Ca2’ bridging is thwarted. This implies a restricted mobility of the long-chain anions in the surface and, furthermore, precludes any phase separation of the sulphonate since this would have concomitant Ca” bridging with flcaz+ 0.5 and would not have the observed easy displacement by Na+. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13
E. Rojas and J.M. Tobias, Biochim. Biophys. Acta, 94 (1965) 393-404. T. Seimiya and S. Okhi, Biochim. Biophys. Acta, 298 (1973) 546-561. D.P. Gregory and L. Ginsberg, Biochim. Biophys. Acta, 769 (1984) 238-244. D.P. Gregory, Ph.D. thesis, Council for National Academic Awards, 1982. D.P. Gregory and J. Mingins, to be published. J. Mingins and J.A.G. Taylor, J. Chem. Sot. Faraday Trans. 1, 71 (1975) 11611171. G. Aniansson, J. Phys. Colloid Chem. 55 (1951) 1286-1299. D.P. Gregory, J. Mingins and G.C. Peterson, to be published. D.O. Shah, J. Colloid Interface Sci., 37 (1971) 744. Y. Hendrikx and D. Mari, C.R. Acad. Sci. Paris, 284C (1977) 813. Y. Hendrikx and D. Mari, C.R. Acad. Sci. Paris, 284C (1977) 971. A.K. Jain and R.P.B. Singh, J. Colloid Interface Sci., 81 (1981) 536. J.M. Corkill, J.R. Goodman and J.R. Tate, Trans. Faraday SOC., 60 (1964) 986.
303-307 Amsterdam
303 -
Printed
in The Netherlands
CALCIUM ASSOCIATION WITH ANIONIC LIPIDS: MODIFICATION BY UNCHARGED CO-CONSTITUENTS
D.P. GREGORY*,
J. MINGINS
and A.L.
SMITH
Unilever Research Port Sunlight Laboratory, Merseyside L63 3JW (United Kingdom) (Received
6 October
1983;
accepted
Quarry
in final form
Road
25 July
East, Bebington,
Wirral,
1984)
ABSTRACT Calcium ion association to surfactant and phospholipid species has been examined using radio-counting, electrokinetic and ion-displacement experiments. Pure and mixed insoluble, charged monolayers spread at an air/water interface were used as a model membrane system. Over the range of primary charge densities examined (0.5-2.5 nm* per charge) for long-chain sulphonate monolayers, the net wall-charge density in the presence of calcium chloride is compounded of the primary head-group charge and associated Ca*+counterions with co-ions playing an insignificant part. The strongly associated Ca2+ is displaced with difficulty by Na’, is isomorphously displaced by Mg*+ and readily displaced by Fe3+. No calcium association to pure monolayers of the uncharged molecules was observed. By spreading mixed monolayers of varying mole fraction and choosing areas of compression, the primary charge density can be held constant whilst at the same time the local monolayer environment is altered. By such means the binding of Cal+ to long-chain sulphonate is studied in the presence of various surface densities of nonionic, zwitterionic or cationic monolayer molecules. Distinct differences in the form of the binding in the various binary mixtures are observed. Similarly, differences had been seen in our earlier work where the anionic species was phosphatidylserine and was mixed with either cholesterol or phosphatidylcholine. Simple models to interpret these findings will be proposed and the possible implications discussed.
INTRODUCTION
The association of calcium ions with anionic lipids is of importance in both surfactant and biological systems. Divalent counterions adsorb strongly at ionic species though there are uncertainties both in the statistics for site binding and the distribution of sites. Examination of the literature reveals no detailed investigation of the number of sites needed nor the influence of the distribution of the sites on the binding. In biological systems, the role of calcium-phosphatidylserine (PS) interaction has prompted studies in model membrane systems. Whilst there have been many studies of the binding of *To
whom
correspondence
0166-6622/85/$03,30
should
0 1985
be addressed.
Elsevier
Science
Publishers
B.V.
304
Ca” to charged tems by charged niques.
PS ]I, 21, the possible modification of the Ca2’ binding by unspecies has received less attention. This paper deals with such sysexamining mixed insoluble monolayers, comprising anionic and unspecies, by radiotracer, surface-pressure and electrokinetic tech-
EXPERIMENTAL
The lipids used were the same samples as described elsewhere [3--51 but were of the highest purity available and in general further purified by HPLC. Inorganic chemicals and water were also as described elsewhere [6]. The lipids were spread to form insoluble monolayers at the air/water interface in a 25 X 6 X 1 cm PTFE trough. Where mixtures were used they were premixed before spreading. The subphase contained 45Ca2’ (from Radiochemical Centre, Amersham, U.K.) diluted with non-radioactive CaCl, to give a final calcium concentration of 1.0 X 10e4 M. The temperature was 20 + 1” C and the pH was approximately 5.5. Surface-associated radioactivity was measured by a modification of the method of Aniansson [7] using an Actigraph-3 gas-flow counting head linked to an ES1 Nuclear ratemeter (model 5350) whose output drove a pen recorder. The increase in radiocount rate relative to background following spreading of a monolayer could be converted to surface-associated Ca’+ by applying a factor obtained by added small known volumes of the subphase CaCl, solution to a glass plate of known area, followed by drying and counting. During radiocounting, monolayer surface pressures were monitored by the Wilhelmy plate method with zero contact angle, using a Beckman LM 600 microbalance, and the area was varied by compression. Electrokinetic measurements were made either underneath a spread lipid monolayer [8] or by preparing mixed particles of PS with cholesterol [3] and measuring their electrophoretic mobilities using a Rank Mk II apparatus with a four-electrode cylindrical cell (Rank Bras, Bottisham, Cambridge, U.K.). The temperature and pH were the same as in the monolayer experiments. RESULTS
AND DISCUSSION
Figure 1 shows surface-pressure/area (n/A) isotherms for monolayers of pure octadecylsulphonate (C,,SO;) and mixtures of this with octadecanol. It can be seen that there is a condensing effect of the octadecanol on the isotherm as previously reported for similar mixtures [9]. The isotherms for each mole ratio from 100/O down to 34/66 C,, SO;/octadecanol are in all cases more condensed than if the substrate were 10-l M NaCl indicating a greater binding of Ca” over Na”. To quantify this Ca” contribution, radiolabelling of the Ca2’ was used. However, because of the long range of the isotope used (45Ca) the mea-
305
sured calcium uptake arises from both bound and diffuse counterions and will be referred to here as associated calcium. For pure sulphonate monolayers, the associated calcium is linearly dependent on the surface concentration of the lipid over the range examined, namely 0.5-2.5 nm* per charge. Denoting the degree of calcium association as Pcaz + and defining it as the surface number density of Ca*’ divided by the surface density of sulphonate, then the data yield a figure of 0.48 for pca2+ over the range examined. This is close to the expected value of 0.5 for a fully bound calcium with a negligible co-ion contribution at such high wall charges. The effect of various added electrolytes on calcium displacement was examined by adding appropriate amounts of stock electrolyte solution to the substrate. Sodium is effective in reducing calcium association only when present at very high levels (> 10“ M), a result again indicative of high specificity of calcium against sodium in agreement with previous findings [lo, 111. In these experiments, magnesium was isomorphous with calcium but ferric ions at the 10e4 M level reduced the calcium association to almost zero. The effect of a second component on the calcium association was examined by incorporating other insoluble materials into the anionic mono-
02
04
03 A /nd
05
06
molecules’
Fig. 1. n/A isotherms for mixtures of C,,SO;/C,,OH at the air-10-4 M CaCl, interface. pH = 5.5, 2’ = 20°C. The mole fraction of C,,SO; is displayed on all the curves (X 100) together with that of the C,,OH on the more expanded curves. The C,,OH label is missing from the more crowded condensed curves but can be readily calculated by difference.
306
layer and comparing the counts at the same surface number density of C,,SO; for various surface mole ratios achieved by means of different degrees of compression. The monolayers were pre-mixed in a common solvent before spreading. Also all calcium measurements were done below the equilibrium spreading pressure of the mixture under investigation. If the second component is an equimolar amount of a cationic species, then the calcium association is reduced to zero. The radiocounting results for sulphonate mixed with octadecanol are shown in Fig. 2. pcaz+ is again as defined for pure sulphonate monolayer. No calcium association was seen for pure octadecanol monolayers. As the amount of spacer material (alcohol) is increased pcaz+ remains substantially constant (there is evidence for a slight decrease of unknown origin as in some previous work [12, 131) up to about 60% “spacer” content, after which there is an increase consistent with a charge reversal occurring in the surface region. Similar behaviour is found for other spacer materials, e.g., methylstearate and in the related phosphatidylserine/ cholesterol system [3] . If the spacer material is zwitterionic, i.e. uncharged but possessing separated positive and negative charges, then it is found that there is no detectable effect on the calcium association either to sulphonate or PS monolayers. These results suggest that calcium binding to the anionic species in the presence of zwitterion permits participation of the anionic moiety of the zwitterion in balancing the double cationic charge of the calcium whereas,
---t
I
I 025 Mole
050 fraction
I 075 %PH
Fig. 2. Calcium binding ratio @Ca z+ as a function of mole fraction monolayers at the air-lO_“ M CaCl, interface. pH = 5.5, T = 20°C.
of C,,OH
in C,,SO;
307
with alcohol as the spacer molecule, unpaired anionic sites might function separately as calcium binding sites leading to a net positive charge of the monolayer. As more spacer is added to the mixture, the probability of anionic molecules being isolated will increase. A simple statistical model can be used to predict the calcium-associated behaviour with varying spacer content and this shows a general agreement between theory and experiment [4, 51. Some implications of a net positive charge in mixed films containing high levels of nonionic monolayer molecules are: (i) There will be an increased electrical contribution to the surface pressure which should expand the n/A isotherm. Close examination of Fig. 1 shows such an expansion with increasing octadecanol content which is not seen on 10-l M NaCl. (ii) In the absence of spacer, calcium is associated very strongly with the film even when Na’ is present at 10e2M. A film which is net positive would be expected to reduce its free energy by exchange with Na’ to reduce the net wall charge. Radiocounting on such films shows Na’ to displace Ca2+ more readily from these high spacer content systems. (iii) If there is a net positive charge at the surface then this should be reflected in the electrokinetic properties of the surface. In the system examined here, the spacer itself has (for unknown reasons) a substantial negative zeta-potential and this contribution needs to be apportioned to the mixture to extract the anionic/Ca2’ potential. Two systems were examined, sulphonate/octadecanol and PS/cholesterol and in neither case was a net positive potential observed although the zeta-potential is numerically lower in the mixture than in the pure state. Surface-potential measurements may aid in the understanding of the electrical properties of these systems. The model developed (and reported in detail elsewhere [4, 51) to accommodate the binding behaviour requires the gradual isolation of the C,,SO; anions from each other with increasing mole fraction of octadecanol so that Ca2’ bridging is thwarted. This implies a restricted mobility of the long-chain anions in the surface and, furthermore, precludes any phase separation of the sulphonate since this would have concomitant Ca” bridging with flcaz+ 0.5 and would not have the observed easy displacement by Na+. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13
E. Rojas and J.M. Tobias, Biochim. Biophys. Acta, 94 (1965) 393-404. T. Seimiya and S. Okhi, Biochim. Biophys. Acta, 298 (1973) 546-561. D.P. Gregory and L. Ginsberg, Biochim. Biophys. Acta, 769 (1984) 238-244. D.P. Gregory, Ph.D. thesis, Council for National Academic Awards, 1982. D.P. Gregory and J. Mingins, to be published. J. Mingins and J.A.G. Taylor, J. Chem. Sot. Faraday Trans. 1, 71 (1975) 11611171. G. Aniansson, J. Phys. Colloid Chem. 55 (1951) 1286-1299. D.P. Gregory, J. Mingins and G.C. Peterson, to be published. D.O. Shah, J. Colloid Interface Sci., 37 (1971) 744. Y. Hendrikx and D. Mari, C.R. Acad. Sci. Paris, 284C (1977) 813. Y. Hendrikx and D. Mari, C.R. Acad. Sci. Paris, 284C (1977) 971. A.K. Jain and R.P.B. Singh, J. Colloid Interface Sci., 81 (1981) 536. J.M. Corkill, J.R. Goodman and J.R. Tate, Trans. Faraday SOC., 60 (1964) 986.